monoamine neurotransmitters as substrates for novel tick sulfotransferases, homology modeling,...

Published: November 2, 2010

r 2010 American Chemical Society 176 dx.doi.org/10.1021/cb100266g |ACS Chem. Biol. 2011, 6, 176–184

ARTICLES

pubs.acs.org/acschemicalbiology

Monoamine Neurotransmitters as Substrates for Novel TickSulfotransferases, Homology Modeling, Molecular Docking, andEnzyme KineticsEmine Bihter Yalcin,† Hubert Stangl,† Sivakamasundari Pichu,‡ Thomas N. Mather,‡ and Roberta S. King*,†

†Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, and ‡ Center for Vector-Borne Disease, University ofRhode Island, Kingston, Rhode Island 02881, United States

bS Supporting Information

ABSTRACT: Blacklegged ticks (Ixodes scapularis) transmit thecausative agent of Lyme disease in the Northeastern UnitedStates. Current research focuses on elucidating biochemicalpathways that may be disrupted to prevent pathogen transmis-sion, thereby preventing disease. Genome screening reportedtranscripts coding for two putative sulfotransferases in wholetick extracts of the nymphal and larval stages. Sulfotransferasesare known to sulfonate phenolic and alcoholic receptor agonistssuch as 17β-estradiol, thereby inactivating the receptor ligands.We used bioinformatic approaches to predict substrates forIxosc Sult 1 and Ixosc Sult 2 and tested the predictions withbiochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface ofthe ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substratesincluding dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity andcorrect conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic bindingmode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (Km =0.1-0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status.Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Becausedopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.

Blacklegged ticks (Ixodes scapularis) transmit the causativeagent of Lyme disease in the United States. The disease is

most prevalent in the North East and Great Lakes regions, qwith12-92 confirmed cases per 100,000 population in 2008 inthese states (CDC, Division of Vector-Borne Infectious Diseases,http://www.cdc.gov/ncidod/dvbid/lyme/ld_rptdLymeCasesbyState.htm, accessed 2/15/2010). This tick species serves as the vectorfor the pathogen, Borrelia burgdorferi, which is a spirochetebacterium responsible for Lyme disease in humans. Smallanimals such as wild mice, chipmunks, and other wild rodentsprovide the reservoir and seem not to be adversely affected bypresence of the bacterium. The spirochete can be passed tohumans after a tick that has previously fed on an infected smallanimal subsequently feeds on an individual. Blacklegged ticks(also known as deer ticks) have three life stages (larval, nymphal,adult) and feed once at each life stage. A single infected tick cantransmit the pathogen, but only after feeding for greater than about24 h.1,2 Thus, strategies to control incidence of Lyme diseaseinclude methods to discourage or shorten feeding duration.

Successful feeding requires an alternating cycle of host blooddrawn into the tick and tick saliva secreted into the host through a

single buccal canal. The saliva of Ixodid ticks contains manypharmacological substances that overcome host-derived de-fenses including substances that prevent coagulation of bloodat the site, mask pain and itch signals from the site, and suppressthe host immune system.3 These substances are secreted into thetick saliva and subsequently are transmitted into the host foraction against the host defenses. In addition, successful feedingrequires endogenous signaling molecules within the tick to turnon and off salivation and control release of substances into thesaliva. Current research is focused on elucidating biochemicalpathways that may be disrupted to prevent transmission ofpathogen to the host, thereby preventing disease.

As part of a genome screening process, Ribeiro et al.4 identifiedtranscripts coding for two putative sulfotransferases [Ixosc Sult1(Ixodes scapularis sulfotransferase member 1; Uniprot Q4PMA2)and Ixosc Sult 2 (Ixodes scapularis sulfotransferase member 2;Uniprot Q4PMA1)] located in the salivary glands of adult Ixodes

Received: August 31, 2010Accepted: November 2, 2010

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177 dx.doi.org/10.1021/cb100266g |ACS Chem. Biol. 2011, 6, 176–184

ACS Chemical Biology ARTICLES

scapularis and in the whole tick extracts of the nymphal and larvalstages. Relative expression levels of the two sulfotransferaseproteins in the tissue was found to be altered upon feeding oflarval and nymphal stage ticks (Pichu et al., unpublished results),which suggests that the function of the Ixosc Sult 1 and Sult 2 maybe important within the process of tick feeding. On the basisof current knowledge of sulfotransferase function in otherspecies,5-7 these enzymes could function to control (i) bloodclotting in the host, (ii) protein secretion in the tick, or (iii)steroid, neurotransmitter, or thyroid activity in the host or tick.Because any of these activities may be relevant to interruption ofthe tick feeding process or to interruption in the pathogentransfer process, we sought to determine substrates for Ixosc Sult1 and Ixosc Sult 2 using bioinformatics and biochemical ap-proaches. All known sulfotransferases catalyze the transfer of an -SO3

- moiety from the physiological donor, 30-phosphoadeno-sine-50-phosphosulfate (PAPS), to an acceptor nucleophile suchas -OH. This results in formation of a negatively charged sulfatefrom a neutral -OH. On the basis of current knowledge ofsulfotransferase function,5-7 there are three possible substratestructure classifications: (a) macromolecules including sugarresidues of arbohydrates and proteoglycans, (b) tyrosine residuesof proteins and peptides, and (c) small molecule phenols andalcohols. The sulfotransferases catalyzing these activities in otherspecies include two major divisions based on their subcellularlocations: membrane-bound sulfotransferases utilize a- and b-class substrates, whereas cytosolic sulfotransferases utilize c-classsubstrates.

This paper describes comparative analyses of structure andsequence among diverse sulfotransferases with preparation of 3Dmodels of Ixosc Sult 1 and 2 in order to predict ligand binding andthe interaction between the ligand and Ixosc Sult 1 and 2. Ourapproaches include global and local amino acid sequence simi-larity, 3D structure conservation among sulfotransferases, andprotein structure modeling with ligand docking. The predictionswere then tested and confirmed using native enzyme tissuehomogenates. Dopamine was found to be a good substrate forboth Ixosc Sult 1 and Sult 2, whereas (R,S)-octopamine wassulfonated only by Ixosc Sult 1.

’RESULTS AND DISCUSSION

Global Sequence Similarity. Our first approach to bioin-formatic characterization of the black-legged tick sulfotrans-ferases was to conduct multiple protein sequence comparisonsfor global sequence similarity of Ixosc Sult 1 and Sult 2 amongover 500 distinct members of the Sulfotransfer-1 family of Pfamfrom diverse species. As shown in Figure 1a, the sulfotrans-ferases clearly diverge according to their subcellular locationand function. Membrane-bound sulfotransferases group inclads shown on the left side of this circular dendrogram; forinstance, the tyrosine protein sulfotransferases (TPST) andthe glycosaminoglycan sulfotransferases (OST, NDST, chon-droitin ST, CHST). On the right half of the circular dendro-gram group the clads containing the cytosolic sulfotransferasesreferred to most commonly by the abbreviations ST or SULT.The SULT 1, 2, and 3 families (mostly mammalian) are well-characterized and have known substrates including endogen-ous and exogenous phenols,and alcohols. The SULT 4, 5, and6 families are also mostly mammalian but do not yet haveknown substrates. Our results show that Ixosc Sult 1 and Sult 2globally align within the cytosolic sulfotransferase superfamily

and thus are likely to utilize small molecule alcohols or phenolsas substrates. This global sequence similarity analysis alsoindicates that Ixosc Sult 1 and 2 are not closely related to themembrane-bound sulfotransferases and thus are unlikely tocontrol blood clotting in the host or protein secretion in thetick.Phylogenetic Analysis. The global amino acid sequence

analysis also allowed evaluation of the phylogenetic relationshipof Ixosc Sult 1 and Sult 2 within the cytosolic sulfotransferasesuperfamily (Figure 1b). Specifically, the maximum likelihoodtree analysis shows that Ixosc Sult 1 and Sult 2 amino acidsequences are most closely related to the cytosolic sulfotrans-ferases from C. elegans (ceST1, Uniprot q9u2z2_caeel) and C.Brassica (Uniprot a8xgq0_caebr). The bootstrap values for theserelationships are 84 and 91, reflecting good confidence. ceST1has been shown to catalyze sulfonation of a variety of phenols,but no endogenous substrate has yet been identified.8

Figure 1. Phylogenetic relationship of Ixosc Sult 1 and Sult 2 to othersulfotransferases. (a) Full distance tree of protein family Sulfotransfer-1ClustalWþmultiple alignment displayed in circular form. Abbreviationsare defined in the text. (b) Phylogenetic best scoring maximum like-lihood tree showing relation to representative cytosolic sulfotrans-ferases. Numbers indicate bootstrap values from 100 trees. Allproteins are identified by their Uniprot codes. Proteins discussed inthe text are noted with their common abbreviated names and examplesubstrates.




We found it interesting that Ixosc Sult 1 and Sult 2 were moreclosely related phylogenetically to the mammalian sulfotrans-ferases (SULT1, SULT2, and SULT3 families) than to sulfo-transferases from other arthropods and nonmammalian species.In the phylogenetic analysis many nonmammalian cytosolicsulfotransferases located to a separate clad with bootstrap valueof 100 (Figure 1b). A few of these nonmammalian sulfotrans-ferases have been characterized including bmST (Bombyx mori,silkworm, Uniprot a0pcf9_bommo), dmST1-4 (Drosophilamelanogaster, fruitfly), and that from Spodoptera frugiperda (fallarmy worm, Uniprot q26490_spofr). The sulfotransferase iso-lated from Bombyx mori was found to catalyze sulfonation of4-nitrocatechol, although no endogenous substrate has yetbeen identified.9 The Spodoptera frugiperda enzyme sulfonatesretinol.10 The cytosolic sulfotransferases from D. melanogaster(dmST1-4) catalyze sulfonation of vanillin, 1-naphthol, 4-nitrophenol, 4-hydroxyecdysone, and dopamine.11

We also compared overall sequence identity and homology ofIxosc Sult 1 and Sult 2 versus individual proteins from otherspecies after pairwise alignment. As observed previously forsulfotransferases from diverse species, the sequence identitywas in the range of 25-35%, and sequence homology was inthe range of 40-55% over the ca. 300 residue sulfotransferasedomain.Local Sequence Similarity and Motif Regions. After obser-

ving that the global sequence analysis showed the Ixosc Sult 1 andSult 2 to be cytosolic sulfotransferases, local amino acid sequencecomparisons were made. Because all sulfotransferases bindPAPS, the residues folding into the PAPS binding pocket arehighly conserved. We found six conserved motifs spacedthroughout the ∼300 amino acid sulfotransferase domain ofthe cytosolic sulfotransferases (Figure 2c). Motifs 1, 2, 3, and 6are the classic cytosolic sulfotransferase motif regions7,12

previously known to make up a portion of the binding cavity forthe cofactor PAPS and containing the catalytic histidine. Motif 1contains PKxGTTW, which has been referred to as signaturesequence Region I13 and the 50PSB motif.7,12 Motif 2 containsthe absolutely conserved catalytic histidine. Motif 3 containsRNPKDxxVS, which has been referred to as the 30PB motif.7,12

Motif 6 contains RKGxxGDWK, which has been referred to assignature sequence Region IV.13 Phylo-mLogo detected twoadditional motif regions that 3D structure analysis found alsoto locate in the PAPS binding region (motifs 4 and 5). All six localsequence motif regions were highly conserved within Ixosc Sult 1and Sult 2 (Figure 2a).Sulfotransferase Structure Conservation and Integrated

Analysis. Integrated 3D structural and protein sequence anal-ysis of the diverse set of crystallized sulfotransferases was used tofurther predict function of the Ixosc Sult 1 and Sult 2 bycomparison (Figure 2). The sequence regions folding into thePAPS binding pocket were previously identified.7,12 Thus, wespecifically sought to identify regions of sequence that fold intothe substrate binding pocket and to compare these residues inIxosc Sult 1 and Sult 2 sequence with characterized sulfotrans-ferases. Substrate binding pocket residues are difficult to identifyby primary sequence comparisons because they are minimallyconserved across sulfotransferase families. We found that super-imposition of 31 published cytosolic sulfotransferase structuresand integrated analysis with the aligned sequences identifiedsuccinct aligned regions that consistently fold into the substratebinding pockets of the crystallized sulfotransferase structures(Figure 2b, S1-S8). From this information, we predicted thatthe residues listed in Table 1 would contain the residues foldinginto the substrate-binding pocket for Ixosc Sult 1 and Ixosc Sult 2.Protein Modeling and Binding Pocket Characterization.

As described in the Methods section, multiple test homology 3D

Figure 2. Mapping of cytosolic sulfotransferase primary and secondary structure to sequencemotif and ligand binding regions. (a) Ixosc Sult 1 and Sult 2primary sequence aligned with conserved sulfotransferase motif residues. (b) Conserved sulfotransferase secondary protein structure is noted bycylinders (R-helix), arrows (β-strands), and lines (loop/turn). Sequence regions folding to form the PAPS binding pocket are labeled P1-P6 (in redhighlight). Sequence regions folding to form the substrate binding pocket are labeled S1-S8 (in blue highlight). (c) Phylo-mLogo motif regions areshown in detail; the one amino acid letter height corresponds to the relative conservation of the particular amino acid.R-Helix andβ-strand numbering isconsistent with Alalli-Hassani et al.6

Table 1. Residues of Ixosc Sult 1 and 2 predicted to fold into the substrate binding pocket

Protein S1a S2 S3 S4 S5 S6 S7

Ixosc Sult 1 GLKFSKDFT SYSKCGAAFVQ D-YVKWSQATPHLELVGAEA- K-H VSIFLNRQLFL GDMIWG FEETPEEDVDA

Ixosc Sult 2 GVPRSPLYT THRKSGTHWVA D-LEDFVRQAPSITLLGASVS R-H YHHVKTRPHYR GRVGQG HRPLHPEAEED

SULT1A3 GVPLIKYF- TYPKSGTTWVS YVRVPFLEVNDPGEPSGLET- K-H YYHFHRMEKAH GEVSYG YTTVPQELMDHa S1-S7 refer to substrate-binding region numbers, as indicated in Figure 2b. The residues underlined provide charge interaction for dopamine andoctopamine ligands.




structure models were created and evaluated (∼500 of Ixosc Sult1,∼400 of Ixosc Sult 2). After evaluation of all model preparationcriteria, one model each for Ixosc Sult 1 (no. 470) and Sult 2 (no.780) were judged the highest quality models and were chosen forfurther studies. These models were based on the X-ray crystalstructure coordinates of 1LS6 (human SULT1A1). Ligandaccessible cavities of both models (Ixosc Sult 1 and 2) werecalculated in the absence of any ligands. The resulting pocketdemonstrated the characteristic shape14 for cytosolic sulfotrans-ferases, which is composed of a ligand binding region and acofactor binding region connected by a channel through which -SO3 transfer occurs (Figure 3a and b).Electrostatic surface calculation facilitated identification of key

features of the defined cavities. Visualization of the electrostaticcharge surface of the residues surrounding the substrate bindingcavity of Ixosc Sult 1 and Sult 2 homology models revealed aregion with substantial negative charge; this region may provideattractive binding interactions for a positive charge region in aligand (Figure 3c and d). In the case of Ixosc Sult 1, aspartate 229provided the negatively charged side chain, whereas in Ixosc Sult2, Glutamate 245 provided the negatively charged side-chain.The negatively charged region located on the edge of the bindingpocket, ∼10 Å away from the sulfate group of PAPS. Thissuggests that molecules with a basic aliphatic amine functionalgroup “tail”, such as the monoamine neurotransmitters, may bepreferred ligands for the Ixosc sulfotransferases. Specific dopamine

sulfotransferases are known to exist in humans (SULT1A3) andin mice (Sult1d1). The 31 superimposed crystal structurecoordinate sets (see Methods section) contained two structuresof SULT1A3 (PDB 1cjm and 2a3r, highest quality model).Comparison of the Ixosc Sult 1 and Sult 2 models with the crystalstructure coordinates of human SULT1A3 (2a3r) showed thatthe critical amine-interacting residue in SULT1A3 (Glu146)15

nearly superimposed with Asp229 and Glu245 identified visuallyfor Ixosc Sult 1 and Sult 2, respectively. Surprisingly, the Glu146of SULT1A3 was located in substrate-binding-region number 5(S5) of the primary sequence, while the Asp229 and Glu245 ofIxosc Sult 1 and Sult 2 located in substrate-binding-regionnumber 7 (S7) (Table 1). In the 3D structure these two regionsfolded just adjacent to one another. Thus, primary sequencealone seems to provide very limited utility for prediction ofsubstrate selectivity in distant sulfotransferases.Docking of PAPS and Potential Substrates. Molecular

docking studies were used to quantitatively explore ligandbinding preferences on the homology model for each sulfotrans-ferase using Autodock 4 and MGL AutodockTools. First, thecofactor PAPS was docked into each model. The resultingbinding mode of PAPS was consistent with the binding modein the published sulfotransferase crystal structures and placed the50-phosphosulfate in a catalytically competent position relative tothe conserved histidine. Subsequently, potential substrate mole-cules were docked into the PAPS/enzyme complex. Dopamine,

Figure 3. Protein structure modeling for Ixodes scapularis Sult 1 and Sult 2. Homology model of Ixosc Sult 1 (a) and Ixosc Sult 2 (b): yellow closedsurface indicates water-accessible cavity available for ligand binding. Electrostatic charge surface display of exposed binding cavity of Ixosc Sult 1 (c) andIxosc Sult 2 (d). Interior of binding pockets were exposed by dissection. Red color indicates partial negative charge, blue color indicates partial positivecharge, white color indicates neutral charge. Substrate binding cavity region of Ixosc Sult 1 (c) contains distinct red color indicating negatively chargedbinding surface available to the substrate. Molecules in stick form show results from docking experiments (PAPS, dopamine).




octopamine, and serotonin were chosen for docking on the basisof the results of the binding pocket characterization indicatingpresence of negatively charged binding region in the substratebinding pocket. 17β-Estradiol and pregnenolone were chosen fordocking because of their neutral molecular charge and character-istic of being common sulfotransferase substrates. We used adocking strategy that repeatedly searches for low energy bindingconformations and calculates the frequency of each low energybinding mode.16 High frequency denotes greater confidence.Catalytically competent binding modes were defined as those inwhich the docked small molecule (dopamine, octopamine,serotonin, estradiol, or pregnenolone) oriented with the -OHwithin 3-4 Å of the NZ of catalytic histidine and with the oxygenwithin 3-4 Å of the PAPS sulfur atom simultaneously. Dopa-mine consistently docked in catalytic mode with reasonableaffinity to both Ixosc Sult 1 and Sult 2, indicating that itwould be a substrate for either sulfotransferase (Figure 4a and d;Table 2). (R)-Octopamine and (S)-octopamine consistentlydocked as a substrate to Ixosc Sult 1 but not to Ixosc Sult 2,indicating some level of substrate selectivity between the twosulfotransferases (Figure 4b and c; Table 2). Serotonin docked toboth sulfotransferases, but only in binding modes that positioned

the -OH too distant (5-6 Å) from the catalytic atoms forcatalysis to occur. Neither 17β-estradiol nor pregnenolonedocked in a catalytically competent mode.Sulfotransferase Activity in Whole Tick Extracts. In order

to test the computational predictions, the cytosolic (100,000� gsupernatant) fractions of native enzyme preparations were

Figure 4. Binding interactions between Ixosc Sult 1/PAPS complex and docked (a) dopamine, (b) (R)-octopamine, and (c) (S)-octopamine,respectively. Similarly, interactions between (d) Ixosc Sult 2 complex and docked dopamine. PAPS, ligands, and catalytic residues (Ser-Lys-His) aredisplayed in stick form, and other interacting residues are displayed in line form with text labels. Interactions were calculated using Ligand Explorer.

Table 2. Computational prediction of Ixodes scapularissulfotransferase substrate selectivity by molecular docking

predicted Kd (μM)a

docked ligand Ixosc Sult 1 model Ixosc Sult 2 model

dopamine 40 67

(S)-octopamine 71 ndb

(R)-octopamine 76 nd

serotonin nd nd

17β-estradiol nd nd

pregnenolone nd nda Predicted Kd values determined as described in the Methods section.b nd = not dock; indicates ligand did not consistently dock in acatalytically competent binding mode.




incubated with each potential substrate under sulfotransferaseconditions. We utilized whole tick extracts from two life stages ofthe Ixodes scapularis ticks: larval and nymphal. The larval stage issignificant because larval ticks feeding on infected animalsbecome infected with the Lyme disease spirochete bacterium.The nymphal stage is significant because nymphal ticks mostoften transfer the spirochete to human hosts. We utilized ticksthat had been fed (allowed access to laboratory animal host undercontrolled conditions) and ticks that had not been allowed tofeed. The rationale for comparing fed and unfed ticks comes fromour work that will be published separately (Pichu, Mather, andKing, unpublished) that showed that expression levels of IxoscSult 1 and Sult 2 was altered by feeding status. Dopamine wasfound to be a good substrate (Km = 0.1-0.4 μM) for the nativeIxodes scapularis sulfotransferases at larval and nymphal stageregardless of their fed/unfed status (Table 3). Octopamine-sulfonation only was detected after feeding, when the geneexpression data suggests that only Ixosc Sult 1 is present(Pichu et al., unpublished). These results agree with the dockingpredictions that octopamine should be sulfonated only by IxoscSult 1, whereas dopamine should be sulfonated by both Ixosc Sult1 and Sult 2. In agreement with the docking predictions, β-estradiol, pregnenolone, and serotonin were not substrates (at 1or 20 μM) for the native tick sulfotransferases.The significance of these findings relates to the known role of

dopamine in stimulation of the salivation signal in ticks. Dopa-mine binds to a dopamine D1 receptor and stimulates salivasecretion in ticks through the activation of adenylyl cyclase andformation of intracellular cyclic AMP causing salivary secretion.17

Octopamine also exhibits neuromodulatory functions inarthropods,18 but it is not known if it functions by the samemechanism as dopamine. It is generally accepted that sulfatedligands do not bind to their receptors because of the replacementof neutral hydrogen-bonding -OH with the negatively chargedsulfate.5 To directly test this assumption, we used a system to testfor agonist activity of dopamine-sulfate at the D2 dopaminereceptor by expressing D2R with KIR3 in Xenopus oocytes.19,20

However, even with high levels of D2R expression, dopamine-sulfate (4 nM) did not elicit detectable activation of D2R in anyof the oocytes injected with both D2R and KIR3 cRNA. Incontrast and in the same oocytes, subsequent perfusion of 4 nMdopamine produced robust activation of D2R as measured by anincrease in KIR3 conductance (Supplementary Figure 6). Thisdata supports the conclusion that dopamine-sulfonation in Ixodesscapularis ticks may result in inactivation of salivary secretion.Saliva secretion is an alternating cycle, where ticks draw in hostblood and salivate out excess fluid and pharmacologically activemolecules. Interruption of this cycle may interfere with successfulfeeding and pathogen transmission into host. Taken together,

these results imply that the function(s) of Ixosc Sult 1 and Sult 2in the Ixodid tick salivary gland may include inactivation of thesalivation signal via sulfonation of dopamine and/or octopamine.

’METHODS

Global Sequence Similarity. The Ixosc Sult 1 and Sult 2sequences were compared with a robust set of 768 highly diversesulfotransferases that were identified via the Protein Family databaseusing the Sulfotransfer-1 family21 (http://www.sanger.ac.uk/Software/Pfam/, PF00685, accessed July 27, 2007, downloaded with all gapsremoved). The sulfotransferase domain contains ca. 300 amino acids,and therefore fragments (defined as <200 residues) were discarded;duplicate sequences were also discarded. Multiple amino acid sequencecomparisons of Ixosc Sult 1 and Sult 2 with the remaining 586sulfotransferase sequences were conducted using Accelrys SeqLab(GCG version 11.1). The guide (distance) tree for the multiplesequence alignment was prepared after slow/accurate pairwise align-ments with ClustalWþ (1.0 gap opening penalty, 0.1 gap extensionpenalty, GONNET scoring matrix). The multiple sequence alignmentwas subsequently conducted in ClustalWþ using the BLOSUM scoringmatrix (1.0 gap opening penalty, 0.1 gap extension penalty, 4 residue gapseparation minimum). The distance tree showing the relationship of theset of 586 sulfotransferases was displayed in circular form using theInteractive Tree of Life online tool22 (http://itol.embl.de/).Phylogenetic Analysis. A set of 50 sulfotransferase amino acid

sequences was analyzed for phylogenetic relationships; the set includedrepresentatives from clads containing SULT families 1-6, and the cladmarked arthropod in Figure 1a. Because different alignment methodsand parameters will produce different results, we usedMCoffee23 (serverversion: http://www.tcoffee.org/) to compute and then combine theoutput of several multiple sequence alignment packages (PCMA, POA,MAFFT, Muscle, TCoffee, ClustalW, ProbCons, DialignTX). Theresulting multiple alignment was then further refined by includingX-ray crystal structure information via Expresso (3DCoffee) (availableat http://www.tcoffee.org/).24,25 This server-based software combinessequence-sequence (ClustalW), structure-structure (SAP), and se-quence-structure (Fugue) information to assemble a progressivealignment.26 Expresso was used under the advanced mode with usercontrol of input structures (the coordinates of the superimposed crystalstructures, described in Sulfotransferase structure conservation section).The MCoffee and Expresso (3DCoffee) methods also estimate the localconsistency between the final alignment and the individual alignmentsby measuring the fraction of individual alignments that are consistent foreach residue.

Subsequently, the refined alignment was used to prepare a phyloge-netic maximum likelihood tree with bootstrap support values. The treewas prepared using RAxML (Randomized Axelerated Maximum Like-lihood) rapid bootstrapping and subsequent ML search27 accessedthrough the MSA hub on the web server of the Vital-IT unit at theSwiss Institute of Bioinformatics (http://myhits.vital-it.ch). The best

Table 3. Sulfotransferase activity in cytosolic fractions of larval and nymphal stage Ixodes scapularis whole tick extracts

dopamine octopamine

tissue Vmaxa (pmol min-1 mg-1) Km dopaminea (μM) Km PAPS a (μM) μM νb pmol min-1 mg-1

nymph unfed 25( 5 0.4( 0.5 3.2( 1.8 1, 10 none detected

nymph fed 48( 7 0.2( 0.4 1.1( 0.9 1 6.4

larvae unfed ν = 0.24 pmolmin-1 mg-1 at 10 μM dopamineb not done

larvae fed 30( 6 0.1( 0.5 2.5( 1.7 1 1.8aVmax, Km dopamine, and Km PAPS were calculated after analysis of sulfonation including a range of dopamine (1.48-3.7 μM) and PAPS (2-10 μM)concentrations. Data plots are available in Supporting Information, Figure 5. b ν was calculated after analysis of sulfonation under single PAPSconcentration (4 μM) and limited number of substrate concentrations (1, 10 μM).



scoring maximum likelihood tree (prepared from 100 bootstrappedtrees) was displayed using the Interactive Tree of Life online tool.22

Several flavanoid sulfotransferases from plants were used as the out-group (FSTL arath, F3ST flach, F4ST flach).Local Sequence Similarity and Sequence Motifs. A set of

174 sulfotransferase amino acid sequences (clads containing all mem-bers of cytosolic SULT families 1-6 and clad marked arthropod inFigure 1a) was analyzed for local sequence motifs, and the conservedmotif residues of cytosolic sulfotransferases were determined withPhylo-mLogo28 (http://cg1.iis.sinica.edu.tw/new/phylomlogo/). Phylo-mLogo considers the frequency of every amino acid in each columnand calculates the variable and homogeneous regions of multiplesequence alignments by base frequencies and entropies. The resultingsequence logo diagrams represent informative patterns in a multiplesequence alignment.Sulfotransferase Structure Conservation and Integrated

Analysis. Structural 3D coordinate sets were obtained from theResearch Collaboratory for Structural Bioinformatics Protein Data Bank(RCSB PDB, www.rcsb.org, 36 structures downloaded July 2006) usingthe keyword search “sulfotransferase”.29 The keyword search collectedone additional protein not considered a sulfotransferase (1SUR, PAPSreductase), which was discarded from the analysis. UCSF Chimerasoftware (www.cgl.ucsf.edu/chimera, 6/21/2007 version, win32platform) was used for integrated 3D structural and protein sequenceanalyses.30,31 Using Chimera, each structure was superimposed onto thecoordinates of 1AQU using the MatchMaker feature (alpha-carbon ofeach aligned residue overlaid, iterated by pruning long atom pairs).1AQU, the first published cytosolic sulfotransferase structure, waschosen as the reference. The transposed coordinates of each of the 36superimposed structures, including crystal ligands, were saved and usedfor all further analyses. The advantage of using the Chimera Match-Maker feature for the superimposition step was the pruning feature, thatis, improving the superimposition in an iterative fashion starting with allatoms matched, and each iteration disregarding the most mismatchedatoms and refining the superimposition based on the remaining atompairs. The structural coordinates of the crystal ligands of the super-imposed structures were used to identify the ligand binding regionsacross all of the sulfotransferases. Specifically, residues contributing tosubstrate binding were identified by highlighting all protein atoms within5 Å of any crystal ligand atom (substrate or inhibitor).Protein Modeling and Characterization. Homology Model

Preparation and Evaluation. Because the success of homology model-ing is dependent on the accuracy of the amino acid alignment of templateand model sequence, the highly refined alignment prepared for thephylogenetic analysis was used as the starting point for homologymodeling. All homology models were created using the commercialsoftware Accelrys Discovery Studio (DS) v 2.1, Build HomologyModelsprotocol, which is based on the MODELER algorithm.32 Parametersettings were generally as follows: cut overhangs, true; disulfide bridges,false; cis-prolines, false; additional restraints, false; copy ligands, false;optimization level, none; refine loops, false. Multiple models (ca. 900)were created to test the effect of template choice and variables such as side-chain refinement (CHI-ROTOR),33 loop refinement (LOOPER),34 andCHARMmminimization. In separate experiments, low, medium and highoptimization levels were also tested.

Eachmodel was evaluated on the basis of a number of parameters. TheDSProteinHealth tool provided quantitation of the validity ofmain-chainconformations (Ramachandran), side-chain conformations (rotamerlibrary), peptide planarity, cis-peptides, R-carbon distances, bond lengths,and bond angles. In addition, each model was superimposed on itstemplate and judged for replication of the general sulfotransferase fold.On the basis of these criteria, onemodel each for Ixosc Sult 1 (no. 470) andSult 2 (no. 780) were chosen for further use. For simplicity, these will bereferred to as Ixosc Sult 1 model and Ixosc Sult 2 model.

Binding Pocket Characterization. The binding sites of the Ixosc Sult1 and Sult 2 models were defined with DS on the basis of the cavitiesinside the proteins. These solvent accessible cavities were calculated withan opening site of 10 Å and grid size of 0.7 Å. The analyzed grid pointswere converted to hydrogen atoms and visualized in soft, closed surfaceform by DS.

The electrostatic charge calculation for atoms in the Ixosc Sult 1 andSult 2 models was performed in DS using Delphi with default param-eters. Delphi allows electrostatic potential determination within thewhole protein’s electrostatic field, resulting in more physically realisticcomputational experiments. Because the catalytic region is located in thecore of the folded protein, each protein was visualized as dissected intohalf in order to visualize the electrostatic surface of the residuessurrounding the ligand cavity.

HomologyModel Optimization. The homology model for Ixosc Sult1 was optimized for ligand docking via limited side-chain refinement andminimization in the presence of cofactor PAPS. The side-chain refine-ment module of DS 2.1 optimizes the protein side-chain conformationon the basis of systematic searching of side-chain conformation andCHARMm energy minimization using the ChiRotor algorithm.33 Basedupon the binding pocket characterization results, the following residuesof the Ixosc Sult 1 model were selected for the side-chain refinement:Asp9, Phe10, Asp13, Ala39, Leu118, Asn119, Phe123, Ser207, Tyr208,Arg248, Gly249, and Thr250. Subsequently, PAPS was docked to theside-chain-refined structure, and the structure was minimized in thepresence of docked PAPS as follows. ACHARMm forcefield was appliedto the receptor and PAPS complex, and minimization was carried outwith following settings: Algorithm, Smart Minimizer; Max Steps, 200;rms Gradient, 0.1; Energy Change, 0.0; Implicit Solvent Model, None;Nonbond List Radius, 14.0; Nonbond Higher Cutoff distance, 12.0;Nonbond Lower Cutoff distance, 10.0; Electrostatics: Spherical Cutoff(Kappa, 0.34, Order, 4); Minimization Constraints, None; ApplySHAKE Constraint, False.

On the basis of the binding pocket characterization results, the IxoscSult 2 homology model was used without side-chain refinement. Thus,PAPS was docked directly to the Ixosc Sult 2 model, and the structurewas minimized in the presence of docked PAPS as described for IxoscSult 1.Docking of PAPS and Potential Substrates. Because current

docking protocols can only dock a single ligand at a time, PAPS was firstdocked into the empty Ixosc Sult 1 and 2 model receptors. Thendopamine, (R)-octopamine, (S)-octopamine, 17β-estradiol, pregneno-lone, and serotonin were individually docked as ligands into eachreceptor-PAPS complex. Autodock 435-37 with Autodock Tools1.4.638,16 on a win64 platform was used for all docking experiments.General docking method was as described previously14 includingLamarckian genetic algorithm search method with 2,500,000 evalua-tions, 27,000 generations, and 300 population size. We used the optionin Autodock 4 for a fully flexible ligand with a nonflexible receptor fordocking. The grid box dimensions, based on the identified bindingcavities from DS, were large enough to cover all possible rotations of theligand. Eight separate docking experiments (of 100 runs each) startingfrom different random conformations were conducted. The 800 result-ing docked conformations were clustered, and those within 2.0 Å rmsdwere considered a single reproducible result. Docked clusters werevisualized, binding interactions were assessed, and atomic distancescalculated using Autodock Tools and Discovery Studio. Clusters wereconsidered to be in catalytic position when all of the following condi-tions were met: OH of ligand within 3-4 Å of the nitrogen atom of thecatalytic histidine, OH of ligand within 3-4 Å of sulfur atom of the 50-phosphosulfate of PAPS. Binding affinity (kcal/mol) was calculated inAutodock 4 as the difference in Gibbs free energy between the boundstate and unbound state of the ligand, incorporating van der Waalsinteractions, hydrogen bonding interactions, desolvation energy,



electrostatic interactions, total free energy, and torsional free energy.The dissociation constant (Kd) was calculated by the equation,ΔG = RTln(Kd), where R is the gas constant, and T is 298.15 K.Sulfotransferase Activity in Native Tissue. Tissue Prepara-

tion. All animal studies were approved by the Institutional Animal Careand Use Committee (protocol number AN01-12-014) and were per-formed in accordance with all national and local guidelines and regula-tions. Laboratory-derived Ixodes scapularis larvae and nymphs were usedfor the study. The fed nymphs and larvae were allowed to feed on micefor 48 h,39 while unfed nymphs and larvae from the same lot were kept incontrol conditions for the same time period. The unfed larval tissue wasderived from a pool of 200 whole unfed larvae, which were homogenizedin 0.1 mL of ice-cold 20 mM potassium phosphate pH 7.0, 1 mMdithiothreitol, 1 mM PMSF. The unfed nymphal tissue was derivedfrom a pool of 180 whole unfed nymphs, which were homogenized in0.1 mL of buffer. Whole fed larval (200/0.1 mL) and whole fed nymphal(50/0.1mL) tissues were prepared similarly. The tissue was homogenizedby sonication followed by centrifugal separation (1 h) and collection ofthe cytosolic fraction (100,000� g supernatant). All manipulations wereat 4 �C.Sulfotransferase Assay. Reaction mixtures contained radiolabeled

sulfonyl donor [35S]-30-phosphoadenosine-50-phosphosulfate (PAP35S),substrate in 20 mM potassium phosphate/1 mM EDTA (pH 7.0), tissuecytosol, and 20 mM potassium phosphate with 1 mM EDTA (pH 7.0).40

After timed incubation at 37 �C, reactions were stopped by heating inboiling water for 30 s. After centrifugation at 10,000� g for 1min to pelletthe protein, an aliquot of the reaction mix was injected onto a HypersilDuet C18/SAX column (150 mm� 4.6 mm, 4 μm). Components wereseparated and eluted using a linear salt gradient of 10-50mMammoniumbicarbonate pH 8.0 in 10% acetonitrile. Radiolabel was detected andquantified on a flow scintillation analyzer (Packard Bioscience, 500TRseries) with Perkin-Elmer Ultima Flo-M scintillation cocktail. PAP35S waseluted at 1.9 min, dopamine-sulfate at 2.9 min, and octopamine-sulfate at3.0 min.

Assay conditions were adjusted to ensure initial-rate conditions forthe kinetic experiments (linear with time, linear with protein concentra-tion, less than 20% substrates utilized). Accordingly, the kinetic experi-ments utilized 1.48-3.7 μM dopamine, 2-10 μM PAP35S, 8 mg mL-1

unfed larval cytosol, 0.4mgmL-1 fed larval cytosol, 0.85mgmL-1 unfednymphal cytosol, or 0.2 mg mL- fed nymphal cytosol and wereincubated for 30 min at 37 �C. Product formation was quantified byflow scintillation counting, and rates of sulfonation were calculated aspmol product formed per minute per milligram of cytosolic protein.Kinetic constants (Km dopamine, Km PAPS, Vmax) were determinedusing nonlinear regression fitting to the Michaelis-Menten two-sub-strate equation by the Enzyme Kinetic (Pharmacology) Module ofSigma Plot 11.

’ASSOCIATED CONTENT

bS Supporting Information. This material is available freeof charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*[email protected].

’ACKNOWLEDGMENT

This study was supported by the University of Rhode IslandCouncil for Research (RSK), and by the University of RhodeIsland Foundation (R.S.K., fund no. 5920). E. B. Yalcin waspartially supported by National Institute of Health grant RO1 AI

37230 (TNM). Accelrys Discovery Studio software was madeavailable through the RI-INBRE Bioinformatics Core Facility(NIH P20 RR16457). The authors report no conflicts of interest.

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monoamine neurotransmitters as substrates for novel tick sulfotransferases, homology modeling,...

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